The present invention relates to the production of sequence-specific DNA binding proteins which function as eukaryotic transcription factors, i.e., transcription regulatory proteins. The invention more particularly relates to the generation of multimeric proteins having nucleic acid (i.e., DNA or RNA) binding domains in which the binding domain or protein interaction surface is engineered or modified to be acidic in nature. Such a nucleic acid binding protein having an acidic multimerization domain is capable of regulating the function of a target nucleic acid sequence or gene to which it is bound, thereby acting as potent dominant-negative regulators of gene transcription, and cell growth and proliferation.
Dominant-negative proteins are capable of inhibiting the binding of nucleic acid binding proteins, i.e., DNA binding proteins, such as transcription regulatory proteins, to target DNA sequences to inactivate gene function. (I. Herskowitz, 1987, Nature, 329:219-222).
The basic-region leucine zipper (xe2x80x9cbZIPxe2x80x9d) DNA binding proteins are a family/class of nucleic acid binding proteins, which are eukaryotic transcription regulatory proteins that regulate transcription of genes by binding as dimers to specific DNA sequences. bZIP proteins characteristically possess two domainsxe2x80x94a leucine zipper structural domain and a basic domain that is rich in basic amino acids (C. Vinson et al., 1989, Science, 246:911-916). The two domains are separated by a short segment known as the fork. Two bZIP proteins dimerize by forming a coiled coil region in which the leucine zipper domains dimerize. The basic regions then interact with the major groove of the DNA molecule at a specific DNA sequence site. The binding to DNA stabilizes the dimer. The dimerization and DNA-interaction event regulates eukaryotic gene transcription.
The leucine zipper motif is common to the primary structure of a number of DNA binding proteins, including the yeast transcription factor GCN4, the mammalian transcription factor CCAAT/enhancer-binding protein C/EBP, and the nuclear transforming oncogene products, Fos and Jun, and is characterized by a repeat of leucine amino acids every seven residues (i.e., a heptad repeat); the residues in this region can form amphipathic xcex1-helices. The leucine-rich amphipathic helices interact and form a dimer complex, called a leucine zipper, at the carboxyl terminus (W. H. Landschultz et al., 1988, Science, 240:1759-1764; A. D. Baxevanis and C. R. Vinson, 1993, Curr. Op. Gen. Devel., 3:278-285), such that the dimerization region forms a coiled coil (E. K. O""Shea et al., 1989, Science, 243:538-542).
Another class of DNA binding proteins, which have similarities to the bZIP motif, are the basic-region helix-loop-helix (xe2x80x9cbHLHxe2x80x9d) proteins (C. Murre et al., 1989, Cell, 56:777-783). bHLH proteins are also composed of discrete domains, the structure of which allows them to recognize and interact with specific sequences of DNA. The helix-loop-helix region promotes dimerization through its amphipathic helices in a fashion analogous to that of the leucine zipper region of the bZIP proteins (R. I. Davis et al., 1990, Cell, 60:733-746; A. Voronova and D. Baltimore, 1990, Proc. Natl. Acad. Sci. USA, 87:4722-4726). Nonlimiting examples of hHLH proteins are myc, max, and mad; myc and mad are known to heterodimerize.
The existence of the leucine zipper in the dimerization region of bZIP proteins allows for a high degree of biological control through the formation of both homodimers and heterodimers. For example, heterodimers are known to form between Fos and Jun (D. Bohmann et al., 1987, Science, 238:1386-1392), among members of the ATF/CREB family (T. Hai et al., 1989, Genes Dev., 3:2083-2090), among members of the C/EBP family (Z. Cao et al., 1991, Genes Dev., 5:1538-1552; S. C. Williams et al., 1991, Genes Dev., 5:1553-1567; and C. Roman et al., 1990, Genes Dev., 4:1404-1415), and between members of the ATF/CREB and Fos/Jun families (T. Hai and T. Curran, 1991, Proc. Natl. Acad. Sci. USA, 88:3720-3724). In general, dimerization of bZIP proteins depends upon the ability of both of the individual carboxyl terminal xcex1-helices to line up in correct register with one another and to generate a symmetric coiled coil. This, in turn, places the amino terminal basic regions in a symmetric orientation, thus allowing them to interact with DNA (A. D. Baxevanis and C. R. Vinson, 1993, Curr. Op. Gen. Devel., 3:278-285). It has been shown that the ability of the helices within the coiled coil to find the proper register with respect to one another is controlled inherently by the individual helices themselves, and not by the placement of the basic region with respect to the DNA (W. Pu and K. Struhl, 1993, Nucleic Acids Research, 21:4348-4355). However, it will be appreciated that the generation of a symmetric coiled coil structure is not a mandatory requirement for the interaction of the multimerization or dimerization domains of various types of nucleic acid binding proteins.
The bZIP proteins are highly conserved throughout the eukaryotic kingdom and have been isolated and identified in yeast, plants, and mammals. These proteins mediate a variety of biological processes, including oncogenesis, memory, segmentation, and energy regulation (R. Boussoudan, 1994, Cell, 79:59-68; S. Cordes, and G. Barsh, 1994, Cell, 79:1025-1034; S. McKnight et al., 1989, Genes Dev., 3:2021-2024; and I. Verma, 1986, Trends in Genetics, 2:93-96.). Therefore, the ability to inhibit the activity of those proteins associated with oncogenesis or abnormal cell growth and proliferation, for example, is a desirable goal in the field.
In addition, inhibition of the production or function of other cellular proteins that are detrimental, or that influence unwanted or inappropriate phenotypes, in cells, tissues, and, ultimately, the whole organism, is an aim for practitioners in the art.
Of the nearly 70 bZIP proteins that have been identified to date, (H. Hurst, 1994, Protein Profiles, 1:123-168), most can be categorized into one of five major subfamilies on the basis of their DNA recognition properties and amino acid sequence similarities (P. F. Johnson, 1993, Mol. Cell. Bio., 13:6919-6930). These bZIP subgroups include the AP-1, CREB (CRE binding protein)/ATF, C/EBP, PAR, (proline- and acidic amino acid- rich protein) and plant G-box proteins. The proteins in each subfamily recognize highly similar or identical DNA sites whose consensus sequences are 9- or 10-base pair palindromes composed of two 5-base pair half-sites. Binding sites for the various classes of bZIP proteins may differ either by their half-site sequences or their half-site spacing properties. AP-1 proteins, such as Fos, Jun, and GCN4 (general control of nitrogen and purine metabolism factor-4) bind to a 9-base pair pseudopalindromic sequence that can be viewed as two half-sites that overlap by a single base pair, while the consensus binding sites for the other four families have directly abutted pairs of half-sites (N. B. Haas et al., 1995, Mol. Cell. Biol, 15:1923-1932). In addition, thyrotrophic embryonic factor (TEF), a transcription factor expressed in the developing anterior pituitary gland, and the liver-enriched albumin D box-binding protein (DBP), (C. R. Mueller et al., 1990, Cell, 61 :279-291), have been reported to constitute another class of bZIP proteins (D. W. Drolet et al., 1991, Genes Dev., 5:1739-1753).
bZIP proteins lacking the transactivation domain are naturally occurring dominant negatives that are generally produced by a genetic deletion of the transactivation domain (A. Clark and K. Dougherty, 1993, Biochem J., 296:521-541; P. Descombes and U. Schibler, 1991, Cell, 67:569-579; N. Foulkes et al., 1991, Cell, 64:739-749; and J. Yin et al., 1994, Cell, 79:49-58). These truncated bZIP proteins are able to dimerize and bind to DNA, and if over-expressed, can act as dominant negatives, presumably by competing with the endogenous bZIP protein for its promoter DNA binding site. Accordingly, the truncated bZIP proteins act by mass action to occlude the normal transactivator from the DNA. In addition, it is possible that the deletion of the transactivation domain could also produce a protein having increased, rather than decreased, DNA binding properties. If this were the case, then this type of truncated and naturally occurring dominant negative would not have to be over-expressed to generate particular phenotypes (A. Braiser and A. Kumar, 1994, J. Biol. Chem., 269:10341-10351).
Needed in the art are proteins, expressed and operative in cells, having dominant-negative function to control the transcription of genes or which regulate RNA production and function in a cell. Such expressed proteins can be used for regulating abnormal cell growth in a variety of eukaryotic organisms, including plants, animals, mammals, including humans, insects, microorganisms, and viruses. The present invention provides to the art proteins which can be modified in a particular way to control gene regulation. The particular type of modification may control gene function, for example, to inhibit abnormal or cancer cell growth and proliferation, to inhibit pathogenic diseases caused by microorganisms, particularly eukaryotic microorganisms, such as yeast, and the like, or viruses and may be used as therapeutics for treating pathological diseases and cancer.
The present invention provides multimeric acidically modified nucleic acid (i.e., DNA or RNA) binding proteins, such as transcription regulating proteins, which have been engineered to contain in their multimerization or protein interaction domains at least one amino terminal acidic amino acid residue. The acidic nature of such nucleic acid binding proteins affects the binding of the proteins to other proteins, e.g., forming heterodimers or heteromultimers, and, ultimately the binding of the proteins to a target DNA or RNA sequence or gene. Nucleic acid binding proteins containing an extension of acidic amino acid residues have an extended protein interaction surface or multimerization or dimerization interface. DNA binding proteins are a particular example of nucleic acid binding proteins suitable for acidic modification according to the present invention. RNA binding proteins are also suitable for use in the invention. In accordance with the invention, the acidic nature of the protein increases the stability of heteromultimeric or heterodimeric complexes that are formed.
It is an object of the present invention to provide an acidic extension to a protein-protein interaction surface or dimerization interface to inactivate the function of a cellular protein. In accordance with the invention, proteins which are useful as drugs, inhibitory molecules, or growth-controlling agents or compounds are provided which, when used both in vitro and in vivo, can inhibit the expression and activity of cellular proteins, the effects of which can be harmful, deleterious, and even lethal, to cell growth and survival.
It is another object of the invention to add acidic amino acid residues to create a multimerization or dimerization surface or extension onto a multimeric complex, particularly a protein, polypeptide or peptide having basic regions that bind to nucleic acids, such as DNA or RNA. More particularly, the acidic extension appended onto the multimeric protein can replace the basic region of such proteins to create molecules that regulate and control cell growth.
It is another object of the invention to regulate gene transcription and expression by providing suitable dominant negative mutant molecules having an acidic phenotype that specifically heterodimerize with native proteins to disrupt the normal action of the native proteins in vivo, thereby causing the subsequent inactivation of cellular gene products.
It is another object of the invention to create dominant negative transcription regulatory proteins by extending the protein-protein interaction surface, making it acidic in nature, to inactivate the function of a cellular protein by specifically and stoichiometrically displacing the native protein from its normal binding with DNA and by inhibiting transactivation and, ultimately, gene transcription and protein production.
It is another object of the invention to provide improved, genetically engineered dominant negative transcription factor proteins that stoichiometrically inhibit the DNA binding of DNA binding proteins, such as bZIP and bHLH proteins, and structurally related types of proteins, to limit the pleiotropic effects normally associated with naturally occurring dominant negative over-expression. The dominant negative mutant proteins of the invention are created by the addition of acidic residues the N-terminus of a multimeric protein, most preferably a DNA binding protein, to produce an acidically modified protein that can interact with a normal cellular protein.
It is another object of the invention to provide methods and rationally-designed constructs suitable for producing and expressing the dominant negative proteins described herein for the specific inactivation of cellular gene products and for use in gene therapy techniques.
It is another object of the invention to utilize the acidic extension of the engineered DNA binding proteins to stabilize a variety of different basic regions of proteins and to create robust dominant negative protein members of various families of DNA binding proteins.
It is yet another object of the invention to provide transgenic animals harboring at least one genetically engineered plasmid construct or vector containing a DNA sequence encoding a DNA binding protein that is acidic in nature to control gene expression in a tissue specific manner.
It is a further object of the invention to provide transgenic animals harboring at least one genetically engineered plasmid construct or vector containing a DNA sequence encoding a DNA binding protein that is acidic in nature which behaves as a dominant negative to the wild type protein and provides viable phenotypes to evaluate and assess the in vivo effects of the protein. Such animals may also be used for rational drug design (i.e., with the dominant negatives considered as drugs) and for testing and evaluating additional or supplemental treatments, drugs, therapies, and the like, and for ameliorating or alleviating the produced transgenic phenotypes.
It is another object to provide dimeric and multimeric nucleic acid binding proteins of plant and animal origin and having acidic multimerization domains allowing the nucleic acid binding protein to bind to a target DNA or RNA sequence, i.e., a specific gene, thereby regulating the function of the gene to which it is bound. Such proteins are expressed, as described herein, from expression eukaryotic or prokaryotic vector constructs molecularly engineered to contain isolated DNA sequences encoding a nucleic acid binding protein having an acidically extended dimerization or multimerization domain.
It is a further object of the invention to control the regulation of a gene through the type of acidic modification that is made to a nucleic acid binding protein, thereby providing therapeutic applications where the target gene or DNA or RNA sequence is present in abnormal or diseased cells and tissues and their normal counterparts. The engineered, acidic nucleic acid binding proteins provide tools for use in cancer therapeutics, diseases caused by eukaryotic microorganisms, for example, yeast, protozoans, algae, parasites, or by viruses, as well as tools for drug development, rational drug design, and drug and gene therapies.
Further objects and advantages afforded by the invention will be apparent from the detailed description hereinbelow.